Retrovirus capsids are unusual in that they are produced inside the maturing virion, not in the cytoplasm or the nucleus of the infected cell. Capsid protein is incorporated into the provirion as part of a spherical shell of the Gag polyprotein. After the provirion has budded off, the viral protease dissects Gag into its matrix (MA), capsid (CA), and nucleocapsid (NC) components. Of these, CA eventually forms the virus capsid, housing the RNA, NC, and the replicase. Evidence suggests that a correctly formed core is essential for infectivity. In FY08 we published a study in which cryo-electron tomography was used to visualize mature virions of Rous Sarcoma Virus (RSV), a prototypic alpha-retrovirus. Their cores were found to be highly polymorphic. We also found that RSV virions, like HIV virions, contain unassembled CA subunits; moreover, the fraction of CA that is assembled correlates with core morphology. These observations implied that initiation of capsid assembly is a critical determinant of core morphology. They also indicated that capsid polymorphism is tolerated, provided that a closed shell is produced and the viral genome and associated enzymes are correctly packaged. During FY14, we focused on two sub-projects. One relates capsid assembly and maturation and their implications for infectivity and how maturation may be inhibited by certain compounds. The second addresses the structure and interactions of Rev, the HIV transactivator. 1) Protease inhibitors were the first drugs to be used successfully against HIV. More recently, another class of antivirals has been identified that inhibits maturation differently. The class member is Beviramat (BVM). We investigated its mode of action by using cryo-electron tomography to determine the three-dimensional structure of virions isolated from HIV-infected cells after BVM treatment. We found that BVM-treated virions contain an incomplete shell of protein underlying the viral envelope, with a honeycomb structure similar to the Gag lattice of immature HIV but lacking the innermost layer that is associated with NC protein. These and other related findings were published in 2011. More recently, we have investigated the question of how the biconical capsid inside mature infectious HIV virions is generated from the spherical shell of Gag molecules in the immature non-infectious virion. The majority of evidence suggested that capsids assemble de novo inside maturing virions from dissociated capsid (CA) protein, but the possibility persisted of a displacive pathway in which the CA shell remains assembled but is remodeled. Inhibition of the final cleavage between CA and spacer peptide SP1/SP blocks the production of mature capsids. We investigated whether retention of SP might render CA assembly-incompetent by testing the ability of Rous sarcoma virus (RSV) CA-SP to assemble in vitro into capsids. Capsids were indeed assembled and were indistinguishable from those formed by CA alone, indicating that SP was disordered. We also used cryo-electron tomography to characterize HIV-1 particles produced in the presence of maturation inhibitor PF-46396 or with the cleavage-blocking CA5 mutation. Inhibitor-treated virions have a shell that resembles the CA layer of the immature Gag shell but is less complete. Some CA protein is generated but usually not enough for a mature core to assemble. We propose that inhibitors like PF-46396 bind to the Gag lattice where they deny the protease access to the CA-SP1 cleavage site and prevent the release of CA. CA5 particles, which exhibit no cleavage at the CA-SP1 site, have spheroidal shells with relatively thin walls. It appears that this lattice progresses displacively toward a mature-like state but produces neither conical cores nor infectious virions. These observations support the disassembly-reassembly pathway for core formation (1). 2) Rev is a small regulatory protein that mediates the nuclear export of viral mRNAs, an essential step in the HIV replication cycle. In this process, Rev oligomerizes in association with a structured RNA molecule, the Rev response element. Detailed information on the structure of Rev and on this interaction is essential for the design of antiviral drugs that impede Rev's function. For many years crystallographic studies were hampered by Rev's tendency to aggregate. However, we were able to construct a hybrid monoclonal antibody whose Fab forms a stable complex with Rev, and solve these co-crystals at 3.2 resolution. These results were published during FY 11. Our continuing research on this system has targeted further investigation of the properties of this antibody. Specifically, we have investigated its ability to block Rev oligomerization and inhibit HIV-1 replication. The Fab itself did not have antiviral activity, but when a Tat-derived cell-penetrating peptide was appended, the resulting molecule (FabRev1-Tat) was strongly inhibitory of three different CCR5-tropic HIV-1 isolates. Computational alanine scanning was used to identify key residues in the complementarity-determining regions to guide mutagenesis experiments. Residues in the light chain CDR3 (LCDR3) were assessed to be important. Residues in LCDR3 were mutated, and LCDR3-Tyr(92) was found to be critical for binding to Rev, as judged by surface plasmon resonance and electron microscopy. Peptides corresponding to all six CDR regions were synthesized and tested for Rev binding. Four of the amide-cyclic forms did. Especially cyclic-LCDR3 (LGGYPAASYRTA) had high affinity for Rev and was able to effectively depolymerize Rev filaments, as shown by bot have this ability (2). An additional study into the homomeric interactions of which Rev is capable has been pursued over several years and is now being prepared for publication. In it, we complexed Rev with a single-chain version of the same Fab (scFv). This complex producing crystals in four different space groups. They were all solved and all revealed essentially the same structure, although the crossing angle varies widely. These experiments are being complemented with cryo-EM studies of the hollow helical tubes that Rev assembles into in vitro. These tubes also exhibited a limited polymorphism, mainly affecting tube diameter, that appears to have its origins in the same crossing-angle variations. The main utility of these studies is to characterize intermolecular interactions that Rev engages in in physiologically relevant complexes.